Kinetics of stop codon recognition by release factor 1

Abstract

Recognition of stop codons by class I release factors is a fundamental step in the termination phase of protein synthesis. Since premature termination is costly to the cell, release factors have to efficiently discriminate between stop and sense codons. To understand the mechanism of discrimination between stop and sense codons, we developed a new, pre-steady state kinetic assay to monitor the interaction of RF1 with the ribosome. Our results show that RF1 associates with similar association rate constants with ribosomes programmed with stop or sense codons. However, dissociation of RF1 from sense codons is as much as 3 orders of magnitude faster than from stop codons. Interestingly, the affinity of RF1 for ribosomes programmed with different sense codons does not correlate with the defects in peptide release. Thus, discrimination against sense codons is achieved with both an increase in the dissociation rates and a decrease in the rate of peptide release. These results suggest that sense codons inhibit conformational changes necessary for RF1 to stably bind to the ribosome and catalyze peptide release.

Figure 1. Interaction of RF1 with the ribosome

(A) A kinetic model for RF1 binding to the ribosome followed by hydrolysis of the newly synthesized protein attached to the P site tRNA. Ribosome (grey), mRNA (orange), P site tRNA (black) with attached protein (blue hexagons), and RF1 (purple). (B) Structure of RF1 bound to the ribosome. Ribosome (grey), E site tRNA (yellow), P site tRNA (red), and RF1 (purple). (C) Recognition of the stop codon in the decoding center by RF1. Stop codon U₁A₂A₃ (orange), RF1 residues (purple), and bases in 16S and 23S rRNAs (grey).

Figure 2. Fluorescence assay to monitor RF1 binding

(A) Sequence of four model mRNAs used to measure RF1 binding to stop or sense codons. The Shine-Dalgarno sequence, P-site, and A-site codons are labeled. Changes from the UAA stop codon are shown in bold and underlined. (B) Increase in fluorescence intensity due to RF1 binding. Fluorescence emission scans before (dotted line) and after addition (black line) of RF1 to release complex with a UAA stop codon in the A site. (C) Examples of fluorescence titrations to determine the K_D of RF1 for sense codons: UUU (square), CUC (triangle), and CAA (circle). Data were analyzed by fitting to a quadratic equation (black line) and normalized from zero to one based upon the best-fit line.

Figure 3. Kinetics of RF1 binding to stop and sense codons

(A) Stopped-flow time course of RF1 (2 μM) binding to release complexes (0.25 μM) with UAA (black trace), CAA (green trace), UUU (purple trace), or CUC (orange trace) codon in the A site. The time courses were fit to a single exponential equation (black line) to determine the observed rate of RF 1 binding (k_obs). (B) Concentration-dependence of RF1 binding. Observed rates are plotted versus RF1 concentration for UAA (circle), CAA (diamond), UUU (triangle), and CUC (square). The standard errors from at least three independent experiments are shown. Plots were fit to a linear equation to determine the association (k_on) and dissociation (k_off) rate constants. In the case of UAA, the y-intercept was constrained to be a positive value. (C) Examples of peptide release time course at saturating RF1 concentrations. Peptide release from release complexes with UAA (circle), CAA (diamond), UUU (triangle), or CUC (square) codons in the A site are indicated. The concentration of RF1 was 9 μM for the stop codon and 200 μM for the sense codons. Data were normalized and fit to a single exponential equation (black line) to determine the rate of peptide release (k_release).